This work was supported by the Swiss National Science Foundation Sinergia grant CRSII3 147697, the Stiftung f&#252;r Angewandte Krebsforschung and Z&#252;rich Krebsliga.

1. Preparation of Cytosolic and Nuclear Protein Fraction
<ol>
	<li>Plate 4 x 106 ACC-MESO-4 cells in a T150 cell culture flask grown in RPMI - 1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin (100x), and 2 mM L-Glutamine (200 mM). When cells reach a confluence of 80-90% (~5-5.5 x 106 cells), proceed with protein extraction of nuclear and cytosolic fraction. 
	NOTE: ACC-MESO-4 cell line was obtained from the RIKEN BioResource Centre11.</li>
	<li>Aspirate medium, wash the cells by adding 15 mL of 1x PBS, tilt the plate gently a few times and aspirate the 1x PBS. Add 3 mL of 0.25% Trypsin-EDTA and incubate the culture flask at 37 &#176;C for 3 min. 
	NOTE: Look at the cells under the microscope for detachment; if still attached tap the flask against the palm of a hand 3 times.</li>
	<li>Add 7 mL of RPMI-1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin (100x), and 2 mM L-Glutamine (200 mM), collect the cells in a 15 mL tube, and spin down at 200 x g for 5 min.</li>
	<li>Aspirate medium and resuspend cell pellet with 1.5 mL of 1x PBS, then transfer the cells into a 1.5 mL microcentrifuge tube. Spin down at 500 x g and 4 &#176;C for 5 min. Discard the supernatant. 
	NOTE: From this step onwards, perform all centrifugation steps at 4 &#176;C and keep all reagents on ice.</li>
	<li>Resuspend the cell pellet with ice-cold 1.5 mL of 1x PBS and spin down the cells at 500 x g and 4 &#176;C for 3 min. Discard the supernatant. 
	NOTE: The required amounts of CER I, CER II, and NER reagents are 10, 0.55, and 5 volumes of estimated cell pellet packed volume, respectively; the following protocol assumes a volume of 20 &#181;L. We recommend preparing only the required amount of reagent CER I.</li>
	<li>Add 200 &#181;L of ice-cold cytoplasmic extraction reagent (CER I) supplemented with 2 &#181;L of 1x proteinase inhibitor, e.g., dilute 1 tablet of Protease Inhibitor (EDTA-free) in 500 &#181;L of water to obtain 100x stock. This amount of reagent CER I is enough to lyse 20 &#181;L of a cell pellet packed volume.
	<ol>
		<li>To estimate the cell pellet packed volume, compare the tube containing the cell pellet with a 1.5 mL tube filled with 10, 20, 50 or 100 &#181;L of 1x PBS.</li>
	</ol>
	</li>
	<li>Resuspend the cell pellet by vigorously vortexing the tube for 15 s and incubate the tube on ice for 10 min. Add 11 &#181;L of ice-cold cytoplasmic extraction reagent II (CER II) to the tube, vigorously vortex for 5 s, and incubate 1 min on ice.</li>
	<li>Vortex vigorously for 5 s and centrifuge the tube at 16,000 x g and 4 &#176;C for 5 min. Transfer the supernatant to the clean pre-chilled tube on ice. The supernatant is the cytoplasmic fraction that is further used in the RNA-pulldown experiment.</li>
	<li>Resuspend the remaining pellet in 100 &#181;L of ice-cold nuclear extraction reagent (NER) supplemented with 1&#160;&#181;L of proteinase inhibitor (100x) and vigorously vortex for 15 s. We recommend preparing only the required amount of NER reagent supplemented with protease inhibitors.</li>
	<li>Incubate the sample on ice for 40 min with occasional vortexing for 15 s (every 10 min).</li>
	<li>Centrifuge the tube at 16,000 x g for 10 min. Transfer the supernatant (this is the nuclear protein fraction) to a clean pre-chilled tube.</li>
	<li>Immediately proceed by measuring protein concentration using the bicinchoninic (BCA) method (see Table of Materials).To control for protein purity of each fraction, perform immunoblotting (Figure 1) against &#945;-tubulin (positive detection only in the cytosolic fraction) and Poly ADP-ribose polymerase - PARP (positive detection only in the nuclear fraction). For protein visualization, refer to section 4.</li>
	<li>In order to keep protein activity and their native state, prepare 25 &#181;L of aliquots of cytosolic and nuclear extracts. Snap-freeze these aliquots by placing them in liquid nitrogen for 5 s and store directly at -80 &#176;C.</li>
</ol>
2. Labeling of RNA with Desthiobiotin
<ol>
	<li>Have RNA probes commercially synthesized and HPLC purified. Resuspend with nuclease-free water at 10 &#181;M. 
	NOTE: Probe sequences are listed in Table 1.</li>
	<li>Use 50 pmol of RNA per RNA-pulldown reaction. Thaw and keep all reagents on ice except for PEG 30%, used to label 3&#39; terminus of RNA oligo.</li>
	<li>Transfer 5 &#181;L of 10 &#181;M RNA probes, labeled as CALB2 3&#39; UTR (ARE), CALB2 3&#39; UTR (mtARE), and Unrelated-RNA (IRE), into 0.5 mL thin-wall microcentrifuge tubes and incubate in a PCR machine at 85 &#176;C for 5 min. Place the tubes immediately on ice. 
	NOTE: This step is important, as it promotes the relaxation and accessibility of the RNA probe for labeling.</li>
	<li>For a single 30 &#181;L reaction, add to the RNA-containing tube the following 10 &#181;L mix: 3 &#181;L of nuclease free water, 3 &#181;L of 10x RNA Ligase Reaction Buffer, 1 &#181;L of RNase Inhibitor (40 U/&#181;L), 1 &#181;L of Desthiobiotinylated Cytidine Bisphosphate (1 mM), and 2 &#181;L of T4 RNA ligase (20 U/&#181;L). 
	NOTE: To prepare master mix A for 3 samples in excess (3.2), mix 9.6 &#181;L of nuclease free water, 9.6 &#181;L of 10x RNA Ligase Reaction Buffer, 3.2 &#181;L of RNase Inhibitor (40 U/&#181;L), 3.2 &#181;L of Desthiobiotinylated Cytidine Bisphosphate (1 mM), and 6.4 &#181;L of T4 RNA ligase (20 U/&#181;L).</li>
	<li>Add carefully 15 &#181;L of PEG 30% to the reaction. Use another tip to mix the reaction. Incubate the reaction overnight at 16 &#176;C.</li>
	<li>The next day, prepare the following: 5 M NaCl (fresh/nuclease-free), chloroform: isoamyl alcohol in 24:1 ratio (e.g., per reaction - 96 &#181;L chloroform and 4 &#181;L of isoamyl alcohol), ice-cold 100% ethanol, and ice-cold 70% ethanol.</li>
	<li>Add 70 &#181;L of nuclease-free water to the RNA-labeling reaction tubes. Add 100 &#181;L of chloroform: isoamyl alcohol and vortex briefly. Spin-down at 13,000 x g for 3 min. Carefully remove ONLY the upper phase and transfer to a new nuclease-free 1.5 mL tube; avoid touching the lower phase.</li>
	<li>Add 10 &#181;L of 5 M NaCl, 1 &#181;L of glycogen and 300 &#181;L of ice-cold 100% ethanol. Place the tube at -20 &#176;C for 2 h. 
	NOTE: At this step, the experiment can be continued on the following day.</li>
	<li>Centrifuge at 13,000 x g and 4 &#176;C for 15 min. Carefully discard the supernatant without disturbing the pellet. Add 300 &#181;L of ice-cold 70% ethanol and centrifuge again at 13,000 x g and 4 &#176;C for 5 min.</li>
	<li>Discard the supernatant completely and air-dry the pellet (15 min). Resuspend the pellet in 20 &#181;L of nuclease-free water. 
	NOTE: Proceed with RNA-pulldown on the same day.</li>
	<li>Incubate the RNA at 90 &#176;C for 2 min and place on ice. Place the labelled-RNA on ice during the following step of pre-washing of the streptavidin magnetic beads. 
	NOTE: A longer incubation time may damage the RNA probe.</li>
</ol>
3. RNA-Protein Pulldown
<ol>
	<li>Pre-washing streptavidin-magnetic beads and incubation with desthiobiotin-RNA
	<ol>
		<li>Use 50 &#181;L of streptavidin magnetic beads per 50 pmol RNA. However, this should be optimized depending on the experiment.</li>
		<li>Vortex the tube with streptavidin-magnetic beads for 15 s, and quickly remove 200 &#181;L (master mix; enough for 3+1 reactions) into a clean 1.5 mL safe-lock tube using cut pipette tips. Place the tube on a magnetic stand so that the beads collect at the side of the tube and wait 1 min. Remove the resuspension liquid.</li>
		<li>To wash the beads, remove the tube from the magnetic stand, add 400 &#181;L of 0.1 M NaOH, 0.05 M NaCl solution, and gently pipet up and down several times. Place the tube back on the magnetic stand. Wait 1 min and collect the supernatant. Repeat this step again.</li>
		<li>Wash the beads with 200 &#181;L of 100 mM NaCl. Remove the supernatant.</li>
		<li>Add 200 &#181;L of 20 mM Tris (pH 7.5), resuspend beads by pipetting, place the tube on the magnetic stand, wait for 1 min and remove the supernatant. Repeat the step.</li>
		<li>Remove the tube from the magnetic stand, add 200 &#181;L of 1x RNA Capture buffer, and resuspend streptavidin magnetic beads by briefly vortexing. Remove 50 &#181;L of streptavidin magnetic beads and add to each labeled-RNA tube using a cut pipette tip. Incubate the tubes for 30 min at room temperature on a roller.</li>
	</ol>
	</li>
	<li>Binding of protein to RNA
	<ol>
		<li>Place the tubes into a magnetic stand, wait 1 min, and remove the supernatant.</li>
		<li>Add 50 &#181;L of 20 mM Tris (pH 7.5) to the beads and resuspend by pipetting. Place the tubes into a magnetic stand, wait 1 min, and remove supernatant. Repeat this step.</li>
		<li>Add 100 &#181;L of 1x Protein-RNA binding buffer to the beads and resuspend by pipetting.</li>
		<li>In the meantime, prepare the following mix: 10 &#181;L of 10x Protein-RNA binding buffer, 30 &#181;L of 50% glycerol, 50 &#181;g of cytoplasmic proteins, and nuclease-free water up to 100 &#181;L. 
		NOTE: Prepare master mix B for 3 samples in excess (3.3) as follows: 33 &#181;L of 10x Protein-RNA binding buffer, 99 &#181;L of 50% glycerol, 165 &#181;g of cytoplasmic proteins and nuclease-free water up to 330 &#181;L. Keep the tube of master mix B on ice.</li>
		<li>Place the tubes into magnetic stand, wait 1 min and collect the supernatant. Add 100 &#181;L of master mix B, resuspend by gently pipetting up and down, and avoid creating bubbles. Incubate reaction tubes for 60 min at 4 &#176;C on a roller.</li>
	</ol>
	</li>
	<li>Washing and elution
	<ol>
		<li>Place the tubes into a magnetic stand, collect the supernatant (which is flow through - FT), and transfer into a new tube. 
		NOTE: Keep this FT on ice for later analysis.</li>
		<li>Pipette 100 &#181;L of 1x Wash buffer on the beads, resuspend gently, put back into the magnetic stand, wait 1 min, and discard the supernatant. Repeat this step 2 times.</li>
		<li>Add 40 &#181;L of Elution Buffer to the magnetic beads, mix by pipetting up and down, and incubate at 37 &#176;C on a tube shaker (950 rpm) for 30 min.</li>
		<li>Place the tubes into a magnetic stand, wait 1 min, and collect eluted sample (which is eluate - E). Place on ice and use for downstream analysis. 
		NOTE: At this step, each tested RNA probe consists of a flow-through (FT) and eluate (E) sample.</li>
	</ol>
	</li>
</ol>
4. Polyacrylamide Electrophoresis and Western Blot Analysis
NOTE: See Table 2 for recipes for the buffers used in this section. Perform a Western blot according to the Laemmli method12.
<ol>
	<li>Handcast 12% SDS-PAGE gels (10-wells, 1.5 mm spacer, 66 &#181;L). Prepare a running gel mix: 4 mL of 30% acrylamide-bisacrylamide stock solution, 2.4 mL of Lower Tris buffer, 3.2 mL of dH2O, 9.6 &#181;L of TEMED, 96 &#181;L of 10% ammonium persulfate solution (APS). Prepare stacking gel mix: 3.25 mL of dH2O, 0.5 mL of 30% acrylamide - bisacrylamide stock solution, 1.3 mL of Upper Tris buffer, 10 &#181;L of TEMED, 20 &#181;L of 10% APS.</li>
	<li>Add 16.6 &#181;L of 6x Laemmli buffer (e.g., 14 mL of Tris/HCl/SDS pH 6.8 (Upper Tris buffer), 2 g of SDS, 1.86 g of DTT, 6 mL of glycerol, and 0.012% bromophenol blue; stored at -20 &#176;C) into the FT sample, and 6.6 &#181;L of 6x Laemmli buffer into E sample. Incubate samples for 5 min at 95 &#176;C.</li>
	<li>Load the samples (20 &#181;L of FT and whole Elute ~40 &#181;L) and run them for 30 min at 60 V, continuing with 20 V overnight.</li>
	<li>Using a semi-dry electroblotting system, transfer proteins onto a PVDF membrane for 80 min at 15 V. 
	NOTE: The PVDF membrane must be activated before performing protein transfer. Incubate the membrane in 100% methanol for 1 min, followed by a 2 min incubation in ultrapure water.</li>
	<li>After the protein transfer, incubate the SDS-PAGE gel with Coomassie solution for 3 h, followed by three steps of washing with ultrapure water, each 30 min.</li>
	<li>At the end of protein transfer, let the PVDF membrane dry for 1 h. Reactivate the membrane as indicated in step 4.5. Block the membrane 1 h with 5% BSA in 1x TTBS.</li>
	<li>Incubate the membrane with the first antibody: HuR or mesothelin, both diluted 1:1000 in 5% BSA in 1x TTBS for 4h at room temperature or overnight at 4 &#176;C.</li>
	<li>Wash the membrane three times for 7 min in 1x TTBS, and incubate with rabbit anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:10,000 in 5% BSA, 1x TTBS.</li>
	<li>Visualize proteins using enhanced chemiluminescence and a digital imager.</li>
</ol>

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L., Yong, J., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+and+post-transcriptional+gene+regulation.">RNA-binding proteins and post-transcriptional gene regulation.</a> FEBS Letters. 582 (14), 1977-1986 (2008).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+biological+roles+of+alternative+3'UTRs.">Evolution and biological roles of alternative 3'UTRs.</a> Trends in Cell Biology. 26 (3), 227-237 (2016).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3'+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA Biology. 9 (5), 563-576 (2012).</li><li>von Roretz, C., Di Marco, S., Mazroui, R., Gallouzi, I. 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Magnetic bead technology for better assay development Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf">https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf</a> (2013)</li><li>Usami, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+four+malignant+pleural+mesothelioma+cell+lines+from+Japanese+patients.">Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients.</a> Cancer Science. 97 (5), 387-394 (2006).</li><li>Laemmli, U. 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The authors declare that they have no competing financial interests.

3&#39; UTRs belong to the non-coding genome3, and all non-coding RNAs can interact with proteins in order to exert their function7. When the mammalian genome was found to be pervasively transcribed and produced a significant portion of long noncoding RNAs16, emerging evidences demonstrated that these long-noncoding RNAs function in regulating gene expression as they interact with chromatin-remodeling complexes17. This knowledg...

Gene expression and the final level of the gene product can be tightly regulated by affecting the mRNA stability and mRNA translation rate1. These post-transcriptional regulatory mechanisms are exerted through the interactions of non-coding RNA and/or RNA-binding proteins (RBPs) with targeted mRNA. It is usually the 3&#39; untranslated region of mRNA (3&#39; UTR - belonging to the non-coding portion of the genome2) that contains specific cis-regulatory elements (CRE), which are recognized by trans-acting factors such as miRNA or RBPs3. The best-studied cis-element within the 3&#39; UTR, is the adenine-rich element (ARE) motif, which is recognized by specific AU-binding proteins (AUBP), and, in turn, induces either mRNA degradation/deadenylation (ARE-mediated decay) or mRNA stabilization4.
The size of the 3&#39; UTR of calretinin mRNA (CALB2) is 573 bp long and contains a putative AUUUA pentamer, as predicted by AREsite2, a bioinformatic tool5. Consistent with the presence of a putative ARE motif, the pmirGLO vector-reporter assay demonstrated a stabilization role of this element within CALB2 mRNA6. Finally, the in vitro RNA-pulldown was used to identify the AUBP that stabilizes calretinin mRNA through the ARE motif.
Since all non-coding RNAs interact with proteins7, the in vitro RNA-pulldown is a good way and first-of-choice assay for identifying RNA-interactors to aid in deciphering molecular mechanisms. In this RNA-pulldown method, commercially synthesized RNA probes, which were labeled with a modified form of biotin (desthiobiotin) at the 3&#39; terminus of the RNA strand, were used. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the bound-RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. Such RNA-bound proteins are eluted from the magnetic beads, run through a 12% SDS-PAGE gel, transferred to a membrane, and probed with different antibodies.
In the standard streptavidin-biotin affinity purification procedure, harsh denaturation conditions are required to disrupt the strong irreversible biotin-streptavidin bond to elute the bound proteins8, which could lead to the dissociation of protein complexes. Unlike biotin, desthiobiotin reversibly binds to streptavidin and is competitively displaced with a buffered solution of biotin, allowing for the gentle elution of proteins, and avoiding the isolation of naturally biotinylated molecules9, suggesting that the technique is ideal for isolating native protein complexes under native conditions.
In vitro binding conditions and stringency, which are determined by salt concentration, reducing agents and detergent percentage, should be close to those present in the cellular context in order to identify true in vivo interactions. The binding conditions implemented herein have been previously demonstrated as appropriate for the identification of the HuR as an AU-binding protein10. This approach could save time, since optimization of proper binding conditions can be time-consuming and challenging. In addition, this method could be used as a starting protocol for any RNA-pulldown experiment and can be gradually optimized by changing the concentration of salts and detergents, changing the glycerol percentage, and adding other salts. Moreover, we demonstrated that even a short 25-nucleotide RNA-probe harboring a pentamer ARE-motif could be used to demonstrate interaction with a specific AUBP.

<table border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">NE-PER Nuclear and cytoplasmic extraction kit</td>
			<td style="width:295px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:124px;">78833</td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Protease Inhibitor Tablets, EDTA-free</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:124px;">A32965</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce Magnetic RNA-protein Pull-Down Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20164</td>
			<td>Includes magnetic beads and therefore the kit need to be stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pierce RNA 3&#8217; End Desthiobiotinylation Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20163</td>
			<td style="width:468px;">The kit is a part of the &#34;Pierce Magnetic RNA-protein Pull-Down Kit&#34;, and needs to be stored at -20 &#176;C unlike the pull-down kit (4 &#176;C).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2,&#160; magnetic stand</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">12321D</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:453px;">Anti - HuR (MOUSE) monoclonal antibody</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">-</td>
			<td style="width:468px;">The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&#160; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - &#945; - tubulin (MOUSE) monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td style="width:124px;">8035</td>
			<td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - PARP (RABBIT) Polyclonal antibody</td>
			<td>Cell Signaling</td>
			<td>9542</td>
			<td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - Mesothelin (MOUSE) Monoclonal Antibody&#160;</td>
			<td>Rockland</td>
			<td>200-301-A87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary goat anti-rabbit antibody</td>
			<td style="width:295px;">Cell Signaling</td>
			<td style="width:124px;">7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary rabbit anti-mouse antibody</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A-9044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RPMI - 1640 medium</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FBS - Filtrated Bovine Serum</td>
			<td style="width:295px;">Pan Biotech&#160;</td>
			<td style="width:124px;">P40-37500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin - streptomycin (100x)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">L-Glutamine solution (200 mM)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:295px;">Gibco by Life technologies&#160;</td>
			<td style="width:124px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN 3 Cell</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini Protean System Glass Plates&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &#181;L</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra Cell Casting Modules</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1658050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNaseZap RNase Decontamination Solution</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:453px;">Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td>
			<td style="width:295px;">Roth</td>
			<td style="width:124px;">3029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">20% SDS&#160;</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td>A3942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">PVDF transfer membrane&#160;</td>
			<td style="width:295px;">Perkin Elmer</td>
			<td style="width:124px;">NEF1002</td>
			<td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Trans-Blot SD semi-dry transfer cell&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Clarity Western ECL Substrate</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1705061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FusionFX&#160; Digital Imager</td>
			<td style="width:295px;">Vilber</td>
			<td style="width:124px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNA oligo synthesis</td>
			<td style="width:295px;">Mycrosynth AG, Switzerland</td>
			<td style="width:124px;">-</td>
			<td>the synthesis scale - 0.04 &#181;mol - HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Bovine serum albumin</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Hydrochloric Acid (HCl) 6 M</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td style="width:124px;">182883.1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ultra Pure 1 M Tris, pH 7.5&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Glycerol&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td>17904</td>
			<td>50% sterile aliquots to be stored at -20&#176;C</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:453px;">Pierce Streptavidin magnetic beads&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">88817</td>
			<td style="width:468px;">Please note that these magnetic beads have an average diameter of 1 &#181;m (range from 0.5 - 1.5 &#181;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &#181;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">TEMED</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ammonium persulfate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coomassie G-250</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A3480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminiumsulfate-(14-18)-hydrate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">368458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ortho-phosphoric acid&#160;</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A0637</td>
			<td></td>
		</tr>
	</tbody>
</table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

In this experiment, a 25-nt long fragment of calretinin 3&#39; UTR harboring ARE motif (CALB2 3&#39; UTR (ARE) 25-nt) was used to test whether it binds specifically to the Human-antigen R (HuR) protein, a known mRNA stabilizer. To test the specificity of the ARE element, a 25-nt RNA probe CALB2 3&#39; UTR (mtARE) containing an ARE-motif mutation, which was previously shown to abolish the stabilization effect of the ARE motif, was used6. The third RNA probe represen...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

desthiobiotin-streptavidin-affinity-mediated-purification-rna

Research

JoVE Journal

Cancer Research

7.9K Views.  University Hospital Zurich. Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

Video: Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...